Colonic mucosal associated invariant T cells in Crohn’s disease have a diverse and non-public T cell receptor beta chain repertoire

Objectives Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn’s disease (CD). Methods The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. Results MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin α4β7 in the colon but not in the blood, where they were enriched for α4β7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. Conclusion MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4β7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.


Introduction
Mucosal-Associated Invariant T (MAIT) cells are a predominantly CD8 + [1,2] sub-population of αβ T cells that were first identified for their invariant use of T cell receptor (TCR) alpha genes TRAV1-2 (TCRVα7.2) and TRAJ33 [3].MAIT cells usually represent about 1-10% of circulating CD3 + lymphocytes in humans, and are enriched in mucosal tissues of the lung, liver, genitourinary tract, and gastrointestinal tract [4,5].MAIT cell TCRs do not recognize MHC-bound peptides, but rather riboflavin precursors [6] presented by the genetically nonpolymorphic major histocompatibility complex class-1 related protein 1 (MR1) [7].These riboflavin precursors are not synthesized by mammalian cells but are instead produced by certain bacteria and yeast, many of which are common to the commensal flora of the gastrointestinal tract.In this way MAIT cells can monitor a diverse array of microbiota through their metabolic products [6].Perhaps as a consequence, MAIT cells are central to early antimicrobial immunity at mucosal barriers and specifically have been shown to mount an immune response to Mycobacterium tuberculosis independent of prior infection [8].Circulating MAIT cells are also reduced in individuals with active Mycobacterium tuberculous [8][9][10], Vibrio cholerae O1 [11], as well as Salmonella typhi infections [12], viral infections [1,[13][14][15][16], and severe bacterial infections resulting in sepsis [17].It is hypothesized that MAIT cell depletion from the blood is a direct result of their recruitment to tissues where they combat active infections [8,18].
A similar decrease in peripheral MAIT cells is found in patients with Multiple Sclerosis (MS) [19], systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [20], Sjo ¨gren's syndrome [21], Type 1 [22] and 2 [23,24] Diabetes, celiac disease [25], ulcerative colitis (UC) and Crohn's disease (CD) [26][27][28][29].A reciprocal increase of MAIT cells was observed in the synovial fluid of RA patients [20] and accumulation of MAIT cells in central nervous system lesions and peripheral nerves of MS patients suggesting that MAIT cells are recruited to inflamed tissues in these settings of autoimmunity [19,[30][31][32][33].However, histological reports differ as to whether MAIT cells are increased [28,29] or decreased [26] in the bowel in CD.In two studies, MAIT cells in blood were reported to be more activated in CD, with more Ki67, BTLA, and NKG2D expression [28] as well as more expression of the cytokines TNF and IL-17A [29], suggesting they are also qualitatively more pro-inflammatory in CD.However, in another study, MAIT cells expressed more activated caspases in CD, suggesting they are more prone to apoptosis [26], in which case their elimination could be pathogenic if they normally play an immunoregulatory role, or limit the activity of microbes that may contribute to disease.
CD is an idiopathic inflammatory bowel disease (IBD) whose pathophysiology shares certain features with the immune response to tuberculosis, such as the formation of granulomas in tissue [34] and vulnerability to TNF blockade [35].Indeed, this has led some to hypothesize that an unknown mycobacterial infection could underlie CD [36], as genetic susceptibility factors for CD show striking overlap with those for mycobacterial infections in genome-wide association studies [37].Given their role in mycobacterial infections, MAIT cells may likewise be central to CD immunopathology.
We describe a more detailed analysis of MAIT cells in CD than has previously been published, including both flow cytometry and full genome transcriptome profiling of these cells in the intestinal mucosa.Furthermore, we describe paired TCR alpha and beta chain gene sequences in individual MAIT cells from colon biopsies, paired with TCR beta sequences from autologous blood MAIT cells, to report on the clonality, diversity, and potentially public nature of these semi-invariant cells in the GI tract in health and disease.

Patient specimens
All human cells were obtained from a biorepository at the Benaroya Research Institute, to which deidentified, donors had previously provided written consent and contributed under a protocol authorized by the Virginia Mason/Benaroya Research Institute institutional review board (IRB), in compliance with the Declaration of Helsinki.For transcriptome profiling of intestinal MAIT and CD8 + T cells, frozen vials of colon lamina propria mononuclear cells (LPMC) were obtained, which had previously come from the surgical resections of six CD patients, six UC patients, and six patients with diverticulosis or neoplasia (not IBD) as the indication for surgery.The resected tissue was inflamed in four of the CD patients, and not inflamed in one CD patient and all six patients without IBD.In the sixth CD patient and all six UC patients, both inflamed and uninflamed samples of colon were both available to be analyzed independently.
For flow cytometry analyses of colon MAIT cells, as well as single cell sorting for TCR sequencing, vials containing frozen colonoscopic biopsies were obtained from ten healthy screening colonoscopy recipients and ten CD patients.Biopsies from inflamed and uninflamed colon were retrieved in separate vials for each of the CD patients.Frozen vials of peripheral blood mononuclear cells (PBMC) were also available for analysis from eight of subjects in each of these cohorts.Clinical and demographic details about these donors are presented in S1 Table.

Specimen processing
Surgically resected colon specimens were processed for homogenized lamina propria (LP) cells and frozen as previously described [69].Colonoscopic jumbo forceps biopsies were placed into bovine calf serum (BCS) at the bedside and promptly transported to the laboratory on ice where an equal amount of 14% DMSO in BCS was added for a final concentration of 7% DMSO for cryopreservation.Biopsies were frozen slowly in freezing jars (-70˚C) following transfer into liquid nitrogen storage (-195.8˚C)until use.PBMC were isolated using Lympho-prep™ (Axis-Shield, Oslo, Norway) density gradient, suspended in 7% DMSO in BCS, and frozen in the same manner.

TCR sequencing
Intact biopsies from HC and CD patients (with paired inflamed and non-inflamed colon specimens from the latter only) were thawed and enzymatically digested in RPMI supplemented with HEPES, PSG, BCS, MgCl 2 , CaCl 2 , Collagenase Type 1, and 0.011% DNase for 30 minutes at 37˚C.Cells were then pelleted, resuspended, and mechanically digested by passing the suspension through an 8-gauge needle and filtered through a 100-μum screen.Cells were again pelleted, stained as above, and immediately sorted using the BD FACS Fusion.Live single MAIT cells were sorted into a 96-well plate and their TCR alpha and beta chain amplified using a nested-PCR protocol [70].PCR products were Sanger sequenced by a commercial third party (GeneWiz; now Azenta Life Sciences, Chelmsford, MA) using primers specific to the common region of the TCR alpha or beta gene, and TCR alignment was performed using the IMGT HighV-quest tool.For blood TCR analyses, up to 50,000 MAIT cells were sorted by FACS as above from thawed PBMC.DNA extracted from sorted MAIT cells was then sent for comprehensive TCR beta chain sequencing by a commercial third party (Adaptive Biotechnologies, Seattle, WA), with data thus generated retrieved from and analyzed on the latter's proprietary online ImmunoSEQ Analyser software.

Bulk RNA-seq experiments and analysis
Cells were sorted directly into lysis buffer from the SMART-Seq v4 Ultra Low Input RNA Kit for sequencing (Takara).Cells were then lysed, and cDNA was synthesized and amplified per the manufacture's instruction.After amplification, sequencing libraries were constructed using the NexteraXT DNA sample preparation kit with unique dual indexes (Illumina, San Diego, CA) to generate Illumina-compatible barcoded libraries.Libraries were pooled and quantified using a Qubit Fluorometer (Life Technologies, Carlsbad, CA).Sequencing of pooled libraries was carried out on a NextSeq 2000 sequencer (Illumina) with paired-end 59-base reads, using a NextSeq P2 sequencing kit (Illumina) with a target depth of 5 million reads per sample.
Base calls were processed to FASTQs on BaseSpace (Illumina), and a base call quality-trimming step was applied to remove low-confidence base calls from the ends of reads.Reads were processed using workflows managed on the Galaxy platform.Reads were trimmed by 1 base at the 3 0 end then trimmed from both ends until base calls had a minimum quality score of at least 30.Any remaining adapter sequence was removed as well.To align the trimmed reads, STAR aligner (v2.4.2a) was used with the GRCh38 reference genome and gene annotations from ensembl release 91.Gene counts were generated using HTSeq-count (v0.4.1).Quality metrics were compiled from PICARD (v1.134),FASTQC (v0.11.3),Samtools (v1.2), and HTSeq-count (v0.4.1).
A quality filter was applied to retain libraries in which the fraction of aligned reads examined compared to total FASTQ reads was > 70%, the median coefficient of variation of coverage was less than 0.85, and the library had at least 1 million reads.All analyzed samples passed these quality filters.Non-protein coding genes and genes expressed at less than 1 count per million in fewer than 10% of samples were filtered out.Expression counts were normalized using the TMM algorithm.For differential gene expression analysis, the linear models for microarray data (Limma) R package after Voom transformation was used; this approach either outperforms or is highly concordant with other published methods.Linear models were generated, and donor identity was included as a random effect.For differential gene expression comparisons, genes with a false discovery rate (FDR) of less than or equal to 0.05 and an absolute expression fold-change greater than or equal to 2 were considered differentially expressed.

Statistical analysis
Analyses were preformed with Prism software (GraphPad).Comparisons between groups of flow cytometry data and other continuous variables did not presume a Gaussian distribution.Therefore, analysis of variance used a Kruskal-Wallis test for unpaired data and a Friedman test for paired comparisons.Two-way comparisons were performed using a Mann-Whitney U-test for unpaired comparisons and a Wilcoxon signed-rank test where data points could be paired (e.g. were from the same donor).Specific tests employed are specified in figures and/or their legends.Given the exploratory nature of these analyses, P-values were not adjusted for multiple comparisons.

Transcriptome analysis of MAIT cells in colon and blood
To compare the gene expression of MAIT (CD3 + , CD4 -, CD161 + , TCRVα7.2 + ) cells to that of CD8 + effector (CD3 + , CD4 -, CD8a + , CD45RA -) T cells from which these MAIT cells were excluded, cDNA was analyzed by RNAseq from each of these two populations after they were sorted by FACS from patient specimens.Although MAIT cells in blood can also be identified with MR1 tetramers, these were not employed because we have found tetramers to poorly label T cells from collagenase-treated colon.Thus, a small subpopulation of CD4 + MAIT cells could not be defined by CD161 and TCRVα7.2,due to CD161 also being expressed by Th17 cells which are enriched in the colon.All CD4 + T cells were therefore excluded from these and subsequent analyses of MAIT cells.Cells were sorted from the cryopreserved PBMC of 6 CD patients, 6 UC patients, and 3 patients without IBD, all of whom were undergoing surgical resection of colon.Additionally, from among the above PBMC donors, these two T cell populations were sorted from cryopreserved LPMC isolated from the surgically resected colonic tissue of 1 non-IBD patient (who underwent sigmoid colon resection as part of a rectopexy procedure for rectal prolapse), 1 CD patient with inflamed colon, 1 CD patient with noninflamed colon, 1 CD patient with both inflamed and non-inflamed colon in separate specimens, 1 UC patient with non-inflamed colon, and 3 UC patients with both inflamed and noninflamed colon in separate specimens.Both CD4 -MAIT and CD8 + effector populations from all 27 of the above specimens produced good-quality libraries, resulting in 2.5 to 12 million reads each, with at least 70% gene alignment and a CV of coverage < 0.8 in all cases.Principal component (PC) analyses divided specimens according to location (blood versus colon) in PC1 and PC2 (Fig 1A ), while dividing them by cell type (CD4 -MAIT vs effector CD8 T cells) in PC3 and PC4 (Fig 1B).These cohort sizes were obviously too small to make multivariate comparisons between diseases or the presence of inflammation, so data was pooled for all colon specimens in the subsequent analyses.
Although the MAIT population sequenced probably contained substantial numbers of other contaminating cells, differential gene expression from normalized raw counts from all subjects revealed a number of genes up-or down-regulated in CD4 -MAIT cells relative to other CD8 cells in the blood (Fig 1C ) or intestine (Fig 1D ).A greater difference in gene expression was seen between CD4 -MAIT and other CD8 T cells in the blood than in the colon, both in terms of fold-change and significance, with the greatest difference observed in LTK, a receptor tyrosine kinase with no known ligand.Other genes up-regulated in CD4 -MAIT cells in this comparison encode several markers and factors known to be associated with both MAIT cells and Th17 cells, such as RORC (RORγt), KLRB1 (CD161), CCR6, as well as the MAITassociated gene SLC4A10 (coding for NCBE, a sodium bicarbonate transporter).Components of the receptors for interleukins 18 (IL18R1, IL18RAP), 12 (IL12RB2) and 23 (IL23R) were also expressed more by CD4 -MAIT cells than other CD8 T cells.In contrast, CD4 -MAIT cells in the blood expressed comparatively less of the immunologically relevant CD8B, CCR4, CCR7, VCAM1, KLRK1 (NKG2D), GPR15, IFNG, CXCR3, GZMH and TIGIT genes than other CD8 T cells.
In the colon, more genes were significantly up-then down-regulated in CD4 -MAIT cells relative to other CD8 T cells, with 202 of the up-regulated genes in the colon shared with those up-regulated by CD4 -MAIT cells in the blood (Fig 1E).The most significantly up-regulated colonic CD4 -MAIT gene was NTN4, encoding netrin 4, a laminin-related protein involved in neurite growth and migration, but with no known role in immunology.As in the blood, IL23R was among the more prominent genes differentially expressed in colonic CD4 -MAIT cells (Fig 1E ), and other genes likewise associated with both MAIT cells and Th17 cells (RORC, KLRB1 and CCR6) were again up-regulated, as in blood CD4 -MAIT cells.In contrast to blood, the cytokine receptor genes IL18R1 and IL12RB2 were not significantly more expressed by CD4 -MAIT cells in the colon, although this may simply reflect the smaller sample size of colonic MAIT cell transcriptomes.
Only 25 genes were up-regulated in CD4 -MAIT cells in the colon while being down-regulated in the blood, relative to other CD8 T cells.Of these, three have well-described immunological roles: IL7 (the cytokine interleukin-7), IL2RA (CD25, the alpha chain of the receptor for interleukin-2), and IFNGR2 (a non-ligand-binding beta chain of the gamma interferon receptor).Additionally, a more pronounced MAIT-specific expression of IL1R1 (CD121a, the receptor for interleukin-1) was seen in the colon relative to blood, as was the gene encoding its signal transducing protein, IRAK2.Curiously, genes encoding some of the proteins involved in signal transduction by the B cell receptor (BANK1, BLNK) were also selectively up-regulated in CD4 -MAIT cells in the colon, while another (BLK) was MAIT-specific in both blood and colon.
Full transcriptome analysis is provided in S2 and S3 Tables, and the normalized log 2 counts in S4 Table.

Colonic MAIT cell immunophenotypes
Flow cytometry was performed on colonoscopic biopsies from 10 CD patients on no pharmaceutical treatment for CD (separately sampling both inflamed and uninflamed colonic segments from each) and 10 HC (screening colonoscopy recipients).Peripheral blood lymphocytes were also available for analyses from 16 of these subjects (8 per cohort).
We and others have previously reported that MAIT cells are less common in the blood of CD than HC subjects [28,71].In contrast, colonic CD3 + , CD4 -, CD161 + , TCRVa7.2 + MAIT cells were a greater fraction of CD4 -negative T cells in the colon in CD relative to HC, even if comparing only grossly uninflamed colon segments (P = 0.015, Fig 2A).Indeed, by paired analysis, there was no correlation between inflammation and this frequency of CD4 -MAIT cells in the colons of CD patients (P > 0.99).The presence of inflammation did, however, correlate with less NKG2D expression by CD4 -MAIT cells (Wilcoxon P = 0.004, Fig 2B ), which also runs contrary to what has been described in blood [28,71].Regardless of inflammation and disease, CD4 -MAIT cells from the colon were predominantly CD8 + and CD103 + (Fig 2C and 2D).By mean fluorescent intensity, per-cell CD103 expression by CD103 + cells was higher in colon MAIT cells from Crohn's patients than healthy controls (Mann-Whitney p = 0.0064, Fig 2E), regardless of inflammation (Wilcoxon p = 0.49).As CD103 is the ligand for E-cadherin, which is mostly expressed by the epithelium, it suggests that most intestinal CD4 -MAIT cells, like many mucosal CD8 + T cells, are intra-epithelial lymphocytes (IEL).Indeed, the deepithelialized LP cells of In contrast, CD103 (integrin αEβ7) expression by circulating CD4 -MAIT cells was rare, being significantly less common among circulating CD4 -MAIT cells than other antigen-experienced CD8 + T cells (Fig 3A).Conversely, significantly more CD4 -MAIT cells expressed the gut-homing integrin α4β7 than did any other antigen-experienced T cells, as previously described [7], with no difference between CD and HC cohorts (Fig 3B).

Colonic MAIT cell TCR sequences
To assess the clonality of MAIT cells in the colon, we FACS sorted each of up to 80 individual CD4 -MAIT cells per biopsy into its own well for paired TCR)alpha and beta chain sequencing from the above colonoscopic biopsies.Perhaps because CD161 expression is more ubiquitous in the colon than blood, flow cytometry alone did not produce a pure MAIT population, as less than half of the sorted cells had a canonical MAIT TCR alpha rearrangement of TRAV1-2/ TRAJ12/20/33 [2][3][4]72,73], with TRAJ33 predominating in all (S5 Table ).Therefore, only productive paired TCR rearrangements of TRAV1-2 and TRAJ12/20/33 alpha chains were considered in further clonotype analysis.These alpha chains showed a consensus CDR3 amino acid sequence (CAV[M/R]DSNYQLIW) that was present in a majority of canonical MAIT cells (Fig 4A ), while no clear consensus was evident in the CDR3 sequences of MAIT TCR beta chains.As described in the literature [2][3][4]72,73], we observed a consistent selection of TCR beta variable genes in colonic CD4 -MAIT cells with TRBV6-4 or TRBV20-1 present in over half of CD4 -MAIT TCRs sequenced, as previously noted [3], but regardless of disease or inflammation (Fig 4B).Additionally, in all cohorts we often found distinct cells with the same TCR alpha and beta chains (clonotypes) in a given biopsy, or common to both of two biopsies from different anatomic locations in the same person (i.e. both the inflamed and uninflamed biopsies from a CD patient) (S6 Table and Fig 5).Despite these TCRs being restricted to a finite range of molecular antigens presented by a genetically non-polymorphic molecule (MR1), there were few "public" TCRs seen in more than one person, none of which were from HC.Only two separate pairs of CD patients had any MAIT TCRs in common, indicating that convergent or pathogenic public clonotypes are not common in this disease.
CD4 -MAIT cells were also sorted from the PBMC of these patients, and total TCR beta chain CDR3 sequencing was performed on their DNA (Adaptive Biotechnologies).TCR diversity of circulating CD4 -MAIT cells in IBD was not significantly different in CD compared to HC (Fig 6A).The overlap in TCR repertoires between blood CD4 -MAIT cells was generally low, with little difference in overlap among or between CD and HC cohorts (Fig 6B and 6C).However, because up to 50,000 MAIT cells were analyzed per PBMC sample, we were able to identify 428 unique TCR beta chain CDR3 amino acid sequences that appeared in blood CD4 - MAIT cells from more than one person (S7 Table ), 103˜of which were uniquely found among CD patients, and 39 of which were uniquely found in HC (the remaining 284 sequences being present in at least one person from each cohort).
Approximately half of the unique MAIT TCR beta sequences we found in biopsies were also found in CD4 -MAIT cells sorted from blood, with the vast majority of this overlap being between autologous samples from the same person.However, 13% of colonic CD4 -MAIT TCR beta sequences could also be found in the blood of another person, regardless of whether Frequency and phenotype of colonic CD4 -MAIT cells.(a) CD4 -MAIT cells from colon biopsies were quantified by flow cytometry as a percent of CD4 -negative T cells and found to be more common in biopsies from ten Crohn's disease (CD) patients relative to ten healthy controls (HC), regardless of whether biopsied colon was inflamed (CDIC) or uninflamed (CDNC).The percent of colonic CD4 -MAIT cells expressing NKG2D (b), CD8 (c), or CD103 (d) and the per-cell mean fluorescent intensity (MFI) of CD103 in the latter was quantified by flow cytometry.The number of MAIT cells (as a percentage of CD4 -T cells) (f) and the percent thereof expressing CD103 (g) was compared between whole mucosal biopsies (Bx: Pooled from (a) and (d), respectively) and the de-epithelialized LP samples used in Fig 1 .The percent of colonic CD8 + conventional T cells expressing CD103 (h) or NKG2D (i) on their surfaceis shown for comparison.A Kruskal-Wallis test for variance was performed for each parameter, and if it revealed a P-value less than 0.05, two-way paired (Wilcoxon) or unpaired (Mann-Whitney) non-parametric comparisons were performed as indicated between autologous or allogeneic specimens, respectively.https://doi.org/10.1371/journal.pone.0285918.g002or not both the blood and colon biopsy donor had CD (Table 1).Thus, expanding our search of colonic CD4 -MAIT TCR beta sequences to also include blood revealed more colonic CD4 - MAIT TCR beta CDR3 sequences to be public (S8 Table ), but again did not find enrichment of these within a cohort to suggest pathogenic or protective clonotypes.

Discussion
As their name would imply, mucosal-associated invariant T cells are part of the adaptive immune system at mucosal surfaces, with a function resembling the innate immune system.MAIT cells are known to respond to metabolite antigens produced by bacteria.Thus, as one of the largest mucosal surfaces in the human body, in contact with the highest density and diversity of bacteria in the human body, the intestinal mucosa is an organ whose MAIT cells warrant characterization.Given their similarity to Th17 cells, implicated in autoimmunity, MAIT cells may be of particular importance in spontaneous intestinal inflammation, such as CD.Indeed, two of the most effective treatment strategies for CD involve targeting integrin α4β7 and signaling through the IL-23 receptor, both of which we find highly enriched on CD4 - MAIT cells.We therefore compared gene expression in CD4 -MAIT cells to that of other CD8 + T cells in the intestines, as well as the blood, and compared blood and intestinal CD4 - MAIT surface protein immunophenotypes and TCR sequences in CD patients and HC.
Reciprocal to evidence that MAIT cells are a smaller fraction of T cells in the blood of CD patients, we corroborated evidence that these cells are enriched in their colons [26][27][28][29].This suggests that MAIT cells are being sequestered from the blood to the intestinal mucosa in CD, as has been proposed to happen in the lungs with pulmonary tuberculosis [8][9][10].We found an increased frequency of CD4 -MAIT cells in the intestinal mucosa of CD patients to be present even in uninflamed tissue, suggesting that MAIT cells predispose to inflammation, rather than being a consequence thereof.While this conflicts with an existing report finding MAIT cells to only be enriched in inflamed colon [29], that study pooled data from CD and UC patients, and also was unable to examine biopsies with more than mild inflammation, which could have overlapped with specimens deemed endoscopically uninflamed in our study.Furthermore, it did not exclude CD4 + T cells, from which a high frequency of CD161 + Th17 cells present in the intestinal mucosa [69] could contaminate MAIT cells.In contrast, the fraction of intestinal CD4 -MAIT cells expressing NKG2D was lower in inflamed than uninflamed colon, suggesting that inflammation leads to its down-regulation.This is consistent with IL-15-driven NKG2D expression by memory CD8 T (including MAIT) cells being inhibited by TCR activation [74].While it is possible that a relative lack NKG2D on MAIT cells could be a cause rather than an effect of local intestinal inflammation, this hypothesis would run contrary to the observation that NKG2D blockade reduced CD clinical activity by week 12 in a randomized trial [75].
We also found that the vast majority of mucosal CD4 -MAIT cells express CD103 (integrin αEβ7), which adheres to the E-cadherin molecule expressed by intestinal epithelial cells [76].While IBD has been associated with a lower percentage of dendritic cells [77], CD8 and CD4 T cells [78] and γδ T cells [79] expressing CD103, we saw no such thing in MAIT cells (Fig 2D ), and by fluorescent intensity we actually found a higher per-cell expression of CD103 protein on the CD103+ MAIT cells of IBD biopsies, regardless of inflammation (Fig 2E).Although analyses of histological morphology were outside the scope of this study, the CD103 + phenotype of intestinal MAIT cells (Fig 2D ) and their relative depletion from de-epithelialized colon lamina propria preparations (Fig 2F) suggests that they are intra-epithelial lymphocytes (IEL), in close proximity to the intestinal lumen, and thus the gut microbiome.A thick layer of mucous produced by the intestinal mucosa limits exposure of the gut epithelium to much of the microbiome itself.However, the riboflavin precursors uniquely recognized by MAIT cells are small organic molecules which may more readily diffuse across mucous than an intact microorganism or its protein antigens.Thus, as IEL, MAIT cells would be uniquely suited to monitor the microbiome through its metabolic products.
Although human MAIT cell clonal diversity has been studied previously in the blood [3,72,73,80], little is known about it at the human colonic mucosal barrier, where TCR diversity is much narrower [69].We therefore had hypothesized that intestinal MAIT cells would be pauciclonal.Furthermore, because they are restricted by the genetically non-polymorphic MR1 molecule [7], and recognize a finite range of riboflavin precursors.[6] we hypothesized that such pauciclonality would include a large number of "public" TCR sequences common to multiple individuals.If so, the presence or absence of specific TCR sequences in the colons of CD patients relative to HC would suggest that specific MR1-restricted antigens are either pathogenic or protective, respectively, in IBD.While the antigens recognized by the colonic CD4 - MAIT cells studied in this report are unknown, the consensus amino acid motif of the TCR alpha chain CDR3s we report (CAV[M/R]DSNYQLIW) most resembles a sequence (CAVRDS[N/D]Y[K/Q]L[S/I]) reported for peripheral blood MAIT cells that respond in vitro to Candida albicans antigens [80].Although not known to be related to disease pathogenesis, C. albicans is an immunogen for the anti-Saccharomyces cerevisiae antibody (ASCA) [81] that has been sufficiently associated with CD to serve as a clinical marker of disease since the 1990's [82] and may predict a more aggressive disease course [83].Thus, further comparison of MAIT TCR sequences between CD patients with and without high ASCA titers may reveal an immunophenotypic difference between these IBD patient cohorts.After restricting analyses to T cells containing the canonical alpha chains with which MAIT cells are defined, we found the corresponding beta chains to be surprisingly diverse, with more than half of all colonic CD4 -MAIT cells analyzed containing a unique TCR beta chain CDR3 hypervariable sequence.Of those colonic MAIT sequences found in more than one cell, only a small minority were shared between different individuals (i.e."public"), and never among the HC subjects we analyzed.Those few "public" TCR beta sequences we identified between colon biopsies from different CD patients could suggest potentially pathogenic antigen specificities, but none of them were shared between more than two patients, so any hypothesized pathogenic specificity would not be a widespread characteristic of this disease.In individual CD patients, the same TCR beta sequence could often be found in MAIT cells that were harvested from both the inflamed and uninflamed colon, suggesting that their antigen specificity is not correlated with tissue inflammation.
"Public" TCR beta chain CDR3 sequences were also uncommon in the peripheral blood, but more readily found due to the much larger number of MAIT cells that could be sorted from blood than biopsies.More than twice as many unique public sequences were exclusive to CD patients as were exclusive to HC.However, more than half of all public sequences were seen in both CD and HC cohorts, suggesting that the observed exclusivity was more a consequence of random chance than any proof that pathogenic (i.e.CD-exclusive) or protective (i.e.HC-exclusive) MAIT clonotypes exist.Indeed, many of the unique colonic CD4 -MAIT TCR beta chain CDR3 sequences seen exclusively in CD patients could also be found in the peripheral blood of HC, and vice versa.Given that the diversity of CD4 -MAIT TCR beta chain sequences appears to greatly exceed the diversity of small, organic molecules in the riboflavin synthesis pathway that can be recognized by MAIT cells, categorization of these beta chain sequences by their ability to bind specific MR1-presented antigens may better clarify similarities and differences between CD and HC.Another possibility that explains the vast TCR βchain repertoire is that there is little requirement for a particular β-chain to recognize the same antigen with most of the recognition being provided by the TCR α-chain.
In summary, we evaluated CD4 -MAIT cells from the blood and colon of people with and without CD, by flow cytometry, mRNA expression, and TCR sequencing.We found CD4 - MAIT cells to be concentrated in the colon in CD relative to HC, regardless of whether or not they came from an inflamed colon segment.Indeed, we found that CD4 -MAIT cells in the blood express more of the gut-homing integrin α4β7 than other CD8 T cells, indicating a tropism for the intestinal mucosa.As integrin α4β7 has become a major therapeutic target in the treatment of IBD, it remains to be seen how a response to anti-integrin therapy affects, or may even be predicted by, MAIT cells.Likewise, we report that colonic CD4 -MAIT cells express much more of the IL-23 receptor than do other CD8 T cells.As IL-23 blockade is also now a major treatment strategy for IBD, a study of MAIT cells before and during anti-IL-23 therapy could also identify biomarkers with which to optimize treatment or predict its efficacy.While colonic CD4 -MAIT cells proved far more polymorphic at the TCR/clonal level than anticipated, with few public TCR sequences to correlate with health or disease, we did observe considerable MAIT TCR repertoire overlap between different anatomic locations within an individual, suggesting an individual would likely also show overlap in their MAIT repertoire at different time points.Longitudinally tracking MAIT TCR repertoire over time in an individual, such as before and during anti-integrin or anti-IL-23 therapy, would provide further insight into how IBD therapeutics work, correlate MAIT cell specificity with disease course, and hence reveal novel insights into the pathogenesis of IBD. and inflammation listed in the header.For biopsies in which these sequences were found, the number of cells with a given sequence found in each biopsy is listed.Sums of rows are in the rightmost column, sums of columns are in the bottom row, and the sum of all is in the lower right cell.(XLSX) S7 Table .TCRβ CDR3 sequences that were found in the MAIT cells sorted from the blood of more than one person are listed.The total list is in the left column, those unique to healthy controls (HC) in the second column, those unique to Crohn's disease (CD) patients in the third column, and those found in both HC and CD patients in the right column.(XLSX) S8 Table .The public TCRβ CDR3 sequences found in the MAIT cells isolated from more than one person are listed in the left column.To the right of each sequences is listed the number of Crohn's disease (CD) or healthy control (HC) patients are shown who had that sequence in MAIT cells from their colon or blood.(XLSX)

Fig 1 .
Fig 1. Blood and mucosal CD4 -MAIT cells have a transcriptome distinct from other CD8 T cells.Full transcriptome sequencing was performed on mRNA isolated from CD4 -MAIT or non-MAIT CD8 + T cells sorted from the blood or surgically resected colonic lamina propria of patients with or without IBD as an indication for surgery.Principal component (PC) analysis was performed on transcriptome profiles, separating anatomic source of cells in PCs 1 and 2 (a), and sorted cell phenotype in PCs 3 and 4 (b).Volcano plots of gene transcripts preferentially expressed in CD4 -MAIT cells (green) versus conventional CD8 T cells (red) are shown for cells sorted from blood (c) or colon (d).Dotted lines denote the threshold for significance, and the most differentially expressed genes are labeled by name.(e) Venn diagram showing the number of gene transcripts significantly more ("_Up") or less ("_Down") expressed in CD4 -MAIT cells relative to other CD8 T cells from PBMC ("Blood") and/or colon ("Tissue").(f) Normalized mRNA expression levels of the IL23R gene compared between CD4 -MAIT cells and non-MAIT CD8 + T cells isolated from blood (PBMC) or colon lamina propria (LPMC) by Mann-Whitney test.https://doi.org/10.1371/journal.pone.0285918.g001 Fig 1 contained fewer MAIT cells (Fig 2F) with less CD103 expression (Fig 2G) than in these whole-mucosa biopsies.NKG2D and CD103 expression by conventional CD8 + T cells from these biopsies is shown for comparison (Fig 2H and 2I).

Fig 2 .
Fig 2.Frequency and phenotype of colonic CD4 -MAIT cells.(a) CD4 -MAIT cells from colon biopsies were quantified by flow cytometry as a percent of CD4 -negative T cells and found to be more common in biopsies from ten Crohn's disease (CD) patients relative to ten healthy controls (HC), regardless of whether biopsied colon was inflamed (CDIC) or uninflamed (CDNC).The percent of colonic CD4 -MAIT cells expressing NKG2D (b), CD8 (c), or CD103 (d) and the per-cell mean fluorescent intensity (MFI) of CD103 in the latter was quantified by flow cytometry.The number of MAIT cells (as a percentage of CD4 -T cells) (f) and the percent thereof expressing CD103 (g) was compared between whole mucosal biopsies (Bx: Pooled from (a) and (d), respectively) and the de-epithelialized LP samples used in Fig 1.The percent of colonic CD8 + conventional T cells expressing CD103 (h) or NKG2D (i) on their surfaceis shown for comparison.A Kruskal-Wallis test for variance was performed for each parameter, and if it revealed a P-value less than 0.05, two-way paired (Wilcoxon) or unpaired (Mann-Whitney) non-parametric comparisons were performed as indicated between autologous or allogeneic specimens, respectively.

Fig 3 .
Fig 3. Frequency and phenotype of blood CD4 -MAIT cells.The percent of blood CD4 -MAIT or other T cell subsets (as indicated on x-axes) expressing CD103 (a) or integrin α4β7 (as detected by labeled vedolizumab) (b) on their surface was quantified by flow cytometry.Pooling data from all cohorts, α4β7 expression showed highly significant variance between T cell subsets by paired analysis (Friedman test), with the most expression among CD4 -MAIT cells, which were significantly more α4β7 + (b) and less CD103 + (a) than other CD8 T cells by paired two-way (Wilcoxon) comparison.No differences in CD103 (a) or α4β7 expression (b) were seen between CD (black dots) and HC (open squares).Error bars reflect means and standard deviations.https://doi.org/10.1371/journal.pone.0285918.g003

Fig 4 .
Fig 4. Colonic CD4 -MAIT cell TCR sequences.Full TCR alpha and beta chain gene transcripts were sequenced from individual CD4 -MAIT cells sorted from colon biopsies of the ten CD and ten HC subjects in Fig 2. Cells lacking the canonical MAIT TRAV1.2 sequence from their alpha chain were deemed contaminants and excluded from analyses.(a) Of all the remaining cells, a consensus sequence was observed in the TCR alpha chain CDR3 region.(b) The fraction of these CD4 -MAIT cells expressing different J-regions from the TCR alpha locus and different V, D, and J regions from the TCR beta locus is shown as a pie chart for inflamed or uninflamed colon biopsies from CD patients, or biopsies from healthy screening colonoscopy recipients.https://doi.org/10.1371/journal.pone.0285918.g004

Fig 5 .
Fig 5. Colonic CD4 -MAIT cell clonality and overlap.Each individual CD4 -MAIT cell from Fig 3 with a canonical TRAV1.2 is shown as a point on the radius of this circos plot.Each individual from whom these MAIT cells were sorted is shown as a different color on the outer circle.The inner circle denotes whether MAIT cells were sorted from the colon biopsy of a HC (green) or a biopsy from the inflamed (red) or uninflamed (blue) colon of a CD patient.Any two MAIT cells with the exact same TCR alpha and beta sequences are connected by a thin line, colored to reflect their tissue of origin as in the inner circle (or purple, if cells from inflamed and uninflamed colon biopsies from CD patients have the same TCR).https://doi.org/10.1371/journal.pone.0285918.g005

Fig 6 .
Fig 6.Blood CD4 -MAIT cell diversity and clonal overlap.CD4 -MAIT cells were sorted from the PBMC of eight Crohn's disease (CD) patients or eight healthy controls (HC) at the time of colonoscopy from which they donated biopsies used in Figs 2-4, and their TCR beta chain CDR3 regions were sequenced.(a) The TCR beta repertoire diversity in each sample is plotted on a log scale, and the difference in Simpson's diversity index (in which 1 denotes monoclonality and 0 means every cell has a different TCR beta sequence) between CD and HC samples shows no significance by unpaired non-parametric analysis (Mann-Whitney).(b) The Morisita index of overlap between TCR repertoires (in which 1 denotes complete overlap and 0 denotes no overlap) from each possible combination of any two of the 16 PBMC donors was calculated, and the overlap between every possible pairing is plotted on a log scale.The overlap between any two HC (HC vs HC), any two CD patients (CD vs CD) or any one HC and any one CD patient (HC vs CD) is shown, with unpaired non-parametric analysis (Kruskal-Wallis test) revealing no significant variance.(c) Actual Morisita indices of diversity plotted in (b) for each possible comparison between two CD (red border) and/or HC (blue border) subjects are shown and color coded on a grid.https://doi.org/10.1371/journal.pone.0285918.g006